2D Electrophoresis

2D Electrophoresis Electrophoresis: the movement of ions in an electric field. Electrophoretic techniques exploit the fact that different ions have different mobilities in an electric field and so can be separated by electrophoresis. Where, μ = free electrophoretic mobility, (μ), with units of (cm2 volt-1 sec-1) K = a constant (embodying the Faraday constant and Avogadro's number) q = net charge on the protein (atomic charges/protein molecule) f = frictional coefficient A Guide to Protein Isolation Author: Dennison, Clive. 2002 P 116 Properties of proteins • Hydrophobicity ;Solubility, location, globularity etc. • UV absorbity ; depends on the Tyr and Trp content • Isoelectric point (PI) ; pH at which a molecule has no net charge. • Molecular weight (MW) ; SDS PAGE, protein sequencing PI MW 2D electrophoresis pK1=1.88 pK2=3.65 pK3=9.60 p p pI of aspartate = 1/2(1.88 + 3.65) = 2.77 pK1=2.32 pK2=9.60 pI of Glycine = 1/2(2.32 + 9.60) = 5.96 Enolase (NP_417259 ) 5.3245650.41720 Isoelectric PointMolecular Weight# Phosphates 1 45728.3812 5.25 2 45806.3452 5.19 http://scansite.mit.edu/calc_mw_pi.html • Isoelectric point (PI) ; pH at which a molecule has no net charge. • MW calculation: MW = ΣAAi x MWi Residue Weight Residue Weight A 71.08 M 131.21 C 103.14 N 114.11 D 115.09 P 97.12 E 129.12 Q 128.14 F 147.18 R 156.2 G 57.06 S 87.08 H 137.15 T 101.11 I 113.17 V 99.14 K 128.18 W 186.21 L 113.17 Y 163.18 Amino Acid Residue Weights • Molecular weight (MW) • (+protein modification) 2D electrophoresis • Cell disruption • Protein solubilization • Prefractionation (optional) • Isoelctric focusing (IEF) • Equilibrium of IEF strip • SDS PAGE • Detection of protein spots • Image analysis and Spot picking • Protein spot identification Cell disruption • osmotic lysis • freeze-thaw cycling • detergent lysis • enzymatic lysis of the cell wall • sonication • grinding with (or without) liquid nitrogen with mortar and pestle • high pressure (e.g. French press) • homogenization with glass beads and a bead beater • a rotating blade homogenizator. • Removal and/or inactivation of interfering compounds (protease, salt, lipid, polysaccharide, nucleic acid, etc) No salt 30mM NaCl Salt interference (E. Coli extract) 2D electrophoresis • Cell disruption • Protein solubilization • Prefractionation (optional) • Isoelctric focusing (IEF) • Equilibrium of IEF strip • SDS PAGE • Detection of protein spots • Image analysis and Spot picking • Protein spot identification Protein solubilization • Chaotropes ;urea - efficient in disrupting hydrogen bonds ;thiourea - breaking hydrophobic interactions • Nonionic and/or zwitterionic detergents ; NP-40, Triton X-100 - not very effective in solubilizing very hydrophobic membrane proteins. ; CHAPS, sulfobetaines (e.g. SB 3-10 or ASB 14) – better for hydrophobic proteins • Reducing agents ; DTT, dithioerythritol (DTE), tributylphosphine (TBP), tris(2- carboxyethyl)phosphine (TCEP) [...].. .2D electrophoresis • • • • • • • • • Cell disruption Protein solubilization Prefractionation (optional) Isoelctric focusing (IEF) Equilibrium of IEF strip SDS PAGE Detection of protein spots Image analysis... with time Selected monomers Engineered pH gradient Easy to handle( strong plastic backing) CH2 CH–C–NH–R O R = weakly acidic or basic buffering group Görg, A , Nature 1991 Proteomics 2004, 4, 3665–3685 2D electrophoresis • • • • • • • • • Cell disruption Protein solubilization Prefractionation (optional) Isoelctric focusing (IEF) Equilibrium of IEF strip SDS PAGE Detection of protein spots Image analysis... improves transfer of protein from IEF strip to SDS gel Dithiothreitol (DTT) - reduction of (reformed) disulphide bridges Iodoacetamide - alkylation of proteins Görg et al., Electrophoresis 1987, 1988 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) SDS The SDS molecules interact with the proteins to give rod-like complexes, containing a constant ratio of SDS per mg of protein At this level... Expensive Selective staining dyes Pro-Q Diamond –Phosphoproteins Pro-Q Emerald –Glycoproteins Pro-Q Amber –Transmembrane Proteins Pro-Q Sapphire –Polyhistidine Proteins 2-D Differential In-Gel Electrophoresis (2D DIGE)-pre-labeling of samples GE Healthcare Internal Control No internal control With internal control GE Healthcare Minimal dye labeling • labeled 1-2 % of lysine residue of protein • Dye... range ;3-5 orders of magnitude •Easy to overlay/compare gels •Minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching 2D Electrophoresis • Parallel comparison • provide map of intact proteins which reflects changes in protein expression, isoforms or post translational modification • resolve more than 5000 protein spots . 2D Electrophoresis Electrophoresis: the movement of ions in an electric field. Electrophoretic techniques. which a molecule has no net charge. • Molecular weight (MW) ; SDS PAGE, protein sequencing PI MW 2D electrophoresis pK1=1.88 pK2=3.65 pK3=9.60 p p pI of aspartate = 1/2(1.88 + 3.65) = 2.77 pK1=2.32 pK2=9.60 pI. 113.17 Y 163.18 Amino Acid Residue Weights • Molecular weight (MW) • (+protein modification) 2D electrophoresis • Cell disruption • Protein solubilization • Prefractionation (optional) • Isoelctric